Using a cohort of more than 550 subjects with a wide variety of eosinophilic disorders, ranging from benign eosinophilia to eosinophilic leukemia, we have continued to identify and characterize novel subgroups of patients with eosinophilia. In this regard, we continue to explore surface receptor expression on eosinophils, measures of eosinophil activation and responses to targeted therapies as ways to increase our understanding of the underlying pathophysiologies in diverse groups of patients with HES. In anticipation of a planned clinical trial with a monoclonal antibody to the inhibitory receptor, Siglec-8, in patients with eosinophilic gastritis, we characterized surface and soluble Siglec-8 levels in normal (ND) and eosinophilic (EO) subjects and assessed the efficacy of anti-Siglec-8 antibodies in inducing eosinophil cell death in vitro. Siglec-8 was consistently expressed on eosinophils from ND and EO and did not correlate with absolute eosinophil count (AEC) or disease activity. Soluble Siglec-8 levels were measurable in serum from most donors, unrelated to AEC or Siglec-8 surface expression. Monoclonal chimeric antibodies to Siglec-8 IgG1 and IgG4 were equally effective at inducing eosinophil cell death after overnight IL-5 priming. In contrast, killing of purified eosinophils without IL-5 was only seen in EO subjects, and NK-mediated eosinophil killing was seen only with the afucosylated anti-Siglec-8 IgG1. Finally, treatment of humanized mice with anti-Siglec antibody led to robust depletion of IL-5-induced eosinophilia in vivo. These data support the use of anti-Siglec-8 antibodies as potential therapeutics for patients with eosinophilic disorders (Legrand et al. J Allergy Clin Immunol, in press). Enhanced killing of eosinophils in the presence of IL-5 may lead to increased efficacy in patients with IL-5-driven eosinophilia. Therapy for eosinophilic disorders remains a primary focus of our group. In the past year, we completed two clinical trials of targeted therapy for the treatment of HES and analyzed data from a compassionate use protocol of mepolizumab for the treatment of treatment-refractory, life-threatening HES. The first clinical trial was an open-label study of dexpramipexole (Knopp Biosciences) in patients with steroid-resistant HES. Dexpramipexole was developed for the treatment of amyotrophic lateral sclerosis (ALS). Although it was well tolerated, dexpramipexole failed to show efficacy in a large phase 3 trial for this disease. It did, however, appear to selectively lower blood eosinophils, which prompted a proof-of-concept open-label phase 2 trial in HES. Four of the 10 subjects enrolled in the study were able to taper prednisone to 50% of their pre-treatment dose, and both primary study endpoints were met (Panch et al. Blood 2018). Three subjects continue on dexpramipexole for 10 to 34 months, two of whom have AEC of 0 on dexpramipexole monotherapy. Tissue biopsies in two subjects confirmed resolution of eosinophilia. Adverse events related to drug have been limited to transient insomnia and palpitations in 3 subjects. Although the mechanism of action of dexpramipexole remains a mystery, bone marrow data is consistent with maturational arrest of the eosinophil lineage. The second trial completed this year was a placebo-controlled, double-blind phase 2 trial of benralizumab (MedImmune/Astra Zeneca), an afucosylated antibody to IL-5 receptor alpha that has shown clinical efficacy in patients with eosinophilic asthma. The current trial stemmed, in part, from our prior pre-clinical work examining IL-5 receptor levels in patients with eosinophilia and/or mastocytosis (Wilson et al. J Allergy Clin Immunol 2011). Twenty adults with PDGFRA-negative HES and AEC 1000 cells/mm3 on stable background therapy were enrolled between April 2014 and January 2017 and randomized to receive benralizumab or placebo monthly for 3 months. while on stable background therapy. At week 12, subjects received open-label benralizumab, and AEC was unblinded beginning at week 13. Depending on the results of the week 13 AEC, subjects were eligible to receive monthly benralizumab with tapering of background therapy. Four The study met the primary endpoint with a >50% reduction in AEC in 9/10 subjects who received benralizumab compared to 3/10 who received placebo. Biopsies of affected tissues were performed in 7 subjects and showed resolution of tissue eosinophilia at week 24 (Kuang et al. J Allergy Clin Immunol 2018; 141(2): AB196). The drug has been well-tolerated with only one serious adverse event (a ureteral stone) potentially related to benralizumab. Only two subjects demonstrated a lack of response at 13 weeks. Four subjects relapsed after an initial complete response, but prior to completion of the trial at week 48. Prior studies have demonstrated that anti-IL5 (mepolizumab) therapy has a glucocorticoid (GC)-sparing effect in GC-sensitive HES (Rothenberg, Klion et al. N Engl J Med 2008). To identify predictors of response to mepolizumab in subjects with severe treatment-refractory HES and compare long-term outcomes in these subjects to HES subjects treated with conventional therapies, we performed a retrospective analysis of clinical and laboratory data from 35 HES subjects treated with mepolizumab on a compassionate use protocol and 55 HES subjects on conventional therapy (Kuang et al. J Allergy Clin Immunol Pract 2018). Peak eosinophilia, GC sensitivity, pulmonary involvement, HES clinical subtype and pre-treatment serum IL-5 correlated with mepolizumab response. Despite evidence of more severe disease at baseline, mepolizumab-treated subjects had comparable long-term clinical outcomes to HES subjects treated with conventional therapies, and reported improvements in therapy-related comorbidities. Subjects managed with mepolizumab monotherapy had fewer disease flares than HES subjects on conventional therapies or mepolizumab-treated HES subjects requiring additional HES therapies. This study confirms that mepolizumab is an effective and well-tolerated therapy for HES, but suggests that response is more likely in GC-responsive subjects with idiopathic or overlap forms of HES. In addition to the clinical trials described above, we continue to explore standard therapies for HES. Glucocorticoids (GC) are the mainstay of therapy for a variety of eosinophil-associated disorders, including hypereosinophilic syndromes (HES), although responses are not universal and GC are associated with significant toxicity. Prior to the first therapeutic use of GC, it was observed that the administration of exogenous adrenocorticotropic hormone or hydrocortisone led to a rapid, profound, and transient eosinopenia in humans. Although this is likely an important first step in the clinical response to GC, little is known about the underlying mechanism. To address this, we assessed the global transcriptional response to GC in human eosinophils through RNA sequencing of peripheral blood eosinophils following a single dose of oral prednisone (1 mg/kg) in three healthy subjects with asymptomatic hypereosinophilia. The kinetics of GC-induced eosinopenia was consistent across the three subjects, with the initial decline occurring between 60 and 120 minutes after GC administration. Among the 414 genes differentially expressed in the first hour after GC administration, the most common were genes associated with apoptosis and CXCR4. Upregulation of CXCR4 protein expression on eosinophils was confirmed in vitro and in vivo, suggesting that this surface receptor is key in the early egress of eosinophils from the circulation (Khoury et al. Allergy 2018).